FLUORESCENT pH DETECTOR SYSTEM AND RELATED METHODS

ABSTRACT

Fluorescent pH detector and methods for measuring pH using the fluorescent pH detector.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.11/207,580, filed Aug. 19, 2005, which claims the benefit of U.S. PatentApplication No. 60/602,684, filed Aug. 19, 2004, and U.S. PatentApplication No. 60/674,393, filed Apr. 22, 2005. Each application isexpressly incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a fluorescent pH detector and methodsfor measuring pH using the fluorescent pH detector.

BACKGROUND OF THE INVENTION

Optical sensors (optrodes) for measuring pH are well known. Certainaromatic organic compounds (like phenolphthalein) change color with pHand can be immobilized on solid supports to form “pH paper.” Thesevisual indicators are easy to use, but do not provide a quantitativereading. The color changes can be difficult to distinguish accurately,and can be masked by colored analyte. Fluorescent indicators have alsobeen used as optical sensors. pH Sensitive fluorescent dyes can beimmobilized on solid supports and generally are more sensitive incomparison to the simple color changing (absorbance or reflectancebased) indicators. The improved sensitivity of fluorescent indicatorsallows the solid support to be miniaturized, and this has been used toadvantage in development of fiber optic sensor devices for measuring pH,CO₂, and O₂ parameters in blood.

A specific need in the medical industry exists for accurate pHmeasurement of blood. The pH of blood, or other bodily fluids (pleuraleffusions) can be associated with certain physiologic responsesassociated with pathology. Blood gas analyzers are common critical careinstruments. Depending on storage conditions, the pH of separated bloodcomponents (plasma, platelets) can change rapidly due to off-gassing ofdissolved CO₂ from the enriched venous blood that is collected from adonor. Platelets in particular are metabolically active, and generatelactic acid during storage at 20-22° C. European quality guidelines forplatelets prepared by the “buffycoat method” require pH of storedplatelets to be pH 6.8-7.4 at 37° C. (7.0-7.6 at 22° C.).

Seminaphthofluorescein (SNAFL) compounds and the relatedseminaphthorhodafluor (SNARF) compounds are commercially availableratiometric fluors (Molecular Probes, Inc., Eugene, Oreg.; see, forexample, U.S. Pat. No. 4,945,171) and their synthesis and spectralproperties have been described. These compounds have advantagesincluding long wavelength absorbance that can be efficiently excitedwith LED light sources. Relevant acid/base equilibria and associatedspectral properties are shown below.

Deprotonation of the naphthol structure of SNAFL dyes gives anaphtholate molecule with longer wavelength fluorescence emission. ThepKa is the pH value where the two molecular species form in equalamounts. SNAFL compounds with reactive linker groups that allow theirconjugation to other molecules of interest are also commerciallyavailable.

Various methods have been used to immobilize “ratiometric” dyes to solidsupports for use in fiber optic pH detectors. Carboxynaphthofluorescein(CNF) has been conjugated to aminoethyl-cellulose and this material wasglued to polyester (Mylar) films to make sensing membranes for optrodes.The pKa of this material was 7.41, slightly lower than the free CNF (pKa7.62). The use of tetraethoxysilane to trap CNF in a sol-gel glass thatwas formed on glass cover slips has also been reported. The pKa of thismaterial was 7.46. A 9-chloro substituted SNAFL analog (SNAFL-2) hasbeen reacted with polyvinylamine and the residual amino groups werecrosslinked with a photocrosslinker to form a gel-like coating onacrylic fibers. The pKa of this fiber-optic sensor was 7.14,significantly lower than the published pKa of the free SNAFL compound(pKa˜7.7). This shows that molecular environment and linker structuresurrounding the immobilized dye can alter the performance of a pHdetector.

Despite the advances made in the detection of pH noted above, thereexists a need for improved methods and devices for measuring pH. Thepresent invention seeks to fulfill this need and provides furtherrelated advantages.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for measuring thepH of a sample. The method is useful in measuring the pH of blood andblood products. In one embodiment, the method is useful in measuring thepH of blood or blood products sealed in a vessel. In one embodiment, themethod includes the steps of:

(a) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe physically isolated from thefluorescent species immobilized on the substrate, wherein thefluorescent species immobilized on the substrate is in liquidcommunication with a sample, wherein the excitation light has awavelength sufficient to effect fluorescent emission from thefluorescent species, wherein the fluorescent species exhibits a firstemission intensity at a first emission wavelength and a second emissionintensity at a second emission wavelength, the ratio of the first andsecond emission intensities being dependent on pH; and

(b) measuring the first and second emission intensities to determinedthe pH of the sample.

In the method, the probe is physically isolated from the fluorescentspecies immobilized on the substrate. As used herein, the term“physically isolated” refers to the physical isolation of the probe fromthe sample being interrogated. The probe providing excitation light andreceiving emission light does contact the sample being interrogated. Inthe method of the invention, the sample is in contact (i.e., liquidcommunication) with the substrate-immobilized fluorescent species. Theprobe is isolated from and does not come not physical contact with thesample. The isolation of the probe from the sample is illustrated inFIGS. 3 and 5. In one embodiment, the probe is isolated from thefluorescent species by a window transparent to the excitation light andthe fluorescent emission.

In one embodiment, the probe comprises one or more optical fibers.

In the method, the fluorescent species is a ratiometric fluorescentspecies. In one embodiment, the fluorescent species is selected from anaphthofluorescein compound and a seminaphthorhodamine compound. In oneembodiment, the naphthofluorescein compound is selected from aseminaphthofluorescein compound and a carboxynaphthofluoresceincompound. In one embodiment, the seminaphthofluorescein compound isselected from 5′ (and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-one(also referred to herein as “SNAFL-1”, see FIG. 7A) and2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid (also referred to herein as “EBIO-3”, see FIG. 7E).

In one embodiment, the fluorescent species immobilized on a substratecomprises a conjugate of a fluorescent species and a macromolecule. Inone embodiment, the macromolecule is an albumin. In one embodiment, themacromolecule is a serum albumin. In one embodiment, the macromoleculeis a human serum albumin. In one embodiment, the macromolecule is arecombinant human serum albumin. In one embodiment, the fluorescentspecies immobilized on a substrate comprises a naphthofluorescein/serumalbumin conjugate. In one embodiment, the fluorescent speciesimmobilized on a substrate comprises a seminaphthofluorescein/humanserum albumin conjugate.

As noted above, the method is suitable for measuring the pH of blood orblood products sealed in a vessel. In one embodiment, the fluorescentspecies immobilized on a substrate is introduced into a sealed vessel bya means that preserves the vessel's seal.

In another aspect of the invention, a system for measuring pH isprovided. In one embodiment, the system includes

(a) a light source for exciting a fluorescent species, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) an assembly for receiving the probe, the assembly comprising:

-   -   (i) a housing for receiving the probe, wherein the housing is        adapted for receiving the probe at a first end and terminating        with a window at the second end, the window being transparent to        the excitation and the emission light,    -   (ii) a tip member reversibly connectable to the housing's second        end, wherein the tip member is adapted to receive liquid from a        sample to be measured, and    -   (iii) a fluorescent species immobilized on a substrate        intermediate the tip member and the window, wherein the        fluorescent species immobilized on the substrate is in liquid        communication with the sample during the measurement, and        wherein the window physically isolates the probe member from the        fluorescent species immobilized on the substrate.

In one embodiment, the light source is a light-emitting diode.

In one embodiment, the first and second detectors are photodiodes.

In one embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the housing comprises a tapered tube terminating withthe window.

In one embodiment, the tip member comprises a spike for puncturing asealed vessel.

The fluorescent species useful in the system include those noted abovein regard to the method and described in further detail below. In oneembodiment, the fluorescent species comprises aseminaphthofluorescein/human serum albumin conjugate.

In a further aspect, the invention provides an assembly useful forintroducing a fluorescent species immobilized on a substrate into asample. The assembly is particularly useful for introducing afluorescent species immobilized on a substrate into a sample sealed in avessel. In one embodiment, the assembly includes

(a) a housing having a first open end and a second closed end, whereinthe closed end comprises a window transparent to visible light;

(b) a tip member reversibly connectable to the housing closed end,wherein the tip member is adapted to expose the housing window; and

(c) a fluorescent species immobilized on a substrate intermediate thehousing window and tip member.

The tip member is adapted to expose the housing window. By exposing thehousing window to the environment exterior to the tip member, thefluorescent species immobilized on the substrate intermediate thehousing window and tip member is in liquid communication with the sampleto be measured.

In one embodiment, the housing is tapered. In one embodiment, the tipmember comprises a spike for puncturing a sealed vessel. The fluorescentspecies useful in the assembly include those noted above in regard tothe method and described in further detail below. In one embodiment, thefluorescent species comprises a seminaphthofluorescein/human serumalbumin conjugate.

In another aspect, the invention provides a blood bag or blood productbag that includes the assembly described above.

In a further aspect of the invention, an environment-sensitivefluorophore protein conjugate is provided. In one embodiment, theconjugate includes an environment-sensitive fluorophore covalentlycoupled to an albumin. In one embodiment, the environment-sensitivefluorophore is a pH-sensitive fluorophore, an oxygen-sensitivefluorophore, a nucleic acid-sensitive fluorophore, an ion-sensitivefluorophore, a glucose-sensitive fluorophore, a lipid-sensitivefluorophore, or an enzyme-sensitive fluorophore.

In one embodiment, the environment-sensitive fluorophore is apH-sensitive fluorophore selected from a naphthofluorescein compound anda seminaphthorhodamine compound. In one embodiment, thenaphthofluorescein compound is a seminaphthofluorescein compound or acarboxynaphthofluorescein compound. In one embodiment, theseminaphthofluorescein compound is 5′ (and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneor2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the albumin is a serum albumin. In one embodiment,the albumin is a human serum albumin. In one embodiment, the albumin isa recombinant human serum albumin.

In one embodiment, the environment-sensitive fluorophore is2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid and the albumin is human serum albumin.

It will be appreciated that the method and system of the inventiondescribed above can be modified to include a particularenvironment-sensitive fluorophores to provide methods and systemsspecific to the utility provided by the particular fluorophore.

In another aspect, the invention provides a substrate-immobilizedfluorescent species. In one embodiment, the substrate-immobilizedfluorescent species is a membrane-immobilized fluorescent species. Inone embodiment, the membrane-immobilized fluorescent species is aconjugate of a naphthofluorescein compound and an albumin adhered to amembrane. In one embodiment, the membrane is a microporous membrane. Inone embodiment, the membrane is a nitrocellulose membrane, a membrane ofmixed esters of nitrocellulose and cellulose acetate, a polyethyleneterephthalate membrane, a polycarbonate membrane, and a polyimidemembrane.

In one embodiment, the naphthofluorescein compound is aseminaphthofluorescein compound or a carboxynaphthofluorescein compound.In one embodiment, the naphthofluorescein compound is 5′ (and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneor2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.

In one embodiment, the albumin is a serum albumin. In one embodiment,the albumin is a human serum albumin. In one embodiment, the albumin isa recombinant human serum albumin.

In another aspect of the invention, a method for measuring carbondioxide is provided. In one embodiment, the method includes the stepsof:

(a) irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe physically isolated from thefluorescent species immobilized on the substrate, wherein thefluorescent species immobilized on the substrate is in liquidcommunication with a solution having pH responsiveness to carbon dioxidepresent in a liquid sample, wherein the solution having pHresponsiveness to carbon dioxide is in communication with the liquidsample through a selectively permeable membrane, wherein the excitationlight has a wavelength sufficient to effect fluorescent emission fromthe fluorescent species, wherein the fluorescent species exhibits afirst emission intensity at a first emission wavelength and a secondemission intensity at a second emission wavelength, the ratio of thefirst and second emission intensities being dependent on pH;

(b) measuring the first and second emission intensities to determinedthe pH of the solution having pH responsiveness; and

(c) correlating the pH of the solution having pH responsiveness to thecarbon dioxide level in the sample.

In one embodiment, the probe is physically isolated from the fluorescentspecies immobilized on the substrate by a window transparent to theexcitation light and the fluorescent emission. In one embodiment, theprobe comprises one or more optical fibers.

In one embodiment, the sample comprises blood or a blood product. In oneembodiment, the sample is contained within a sealed vessel. In oneembodiment, the fluorescent species immobilized on a substrate isintroduced into a sealed vessel by a means that preserves the vessel'sseal.

The fluorescent species useful in the method include those noted abovein regard to the method for measuring pH and described in further detailbelow. In one embodiment, the fluorescent species comprises aseminaphthofluorescein/human serum albumin conjugate.

In another aspect, the invention provides a system for measuring carbondioxide. The system is useful for measuring the carbon dioxide level ina liquid sample. In one embodiment, the system includes:

(a) a light source for exciting a fluorescent species, wherein thefluorescent species has a first emission intensity at a first emissionwavelength and a second emission intensity at a second emissionwavelength;

(b) a first emission detector for measuring the first emissionintensity;

(c) a second emission detector for measuring the second emissionintensity;

(d) an excitation lightguide for transmitting excitation light from thelight source to the fluorescent species, wherein the lightguidecomprises a first terminus proximate to the light source and a secondterminus distal to the light source;

(e) a first emission lightguide for transmitting emission from thefluorescent species to the first emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(f) a second emission lightguide for transmitting emission from thefluorescent species to the second emission detector, wherein thelightguide comprises a first terminus proximate to the detector and asecond terminus distal to the detector;

(g) a probe housing the distal termini of the excitation lightguide,first emission lightguide, and second emission light guide; and

(h) an assembly for receiving the probe, the assembly comprising:

-   -   -   (i) a housing for receiving the probe, wherein the housing            is adapted for receiving the probe at a first end and            terminating with a window at the second end, the window            being transparent to the excitation and the emission light,        -   (ii) a tip member reversibly connectable to the housing's            second end, wherein the tip member comprises a chamber for            receiving a solution having pH responsiveness to carbon            dioxide present in a liquid sample, wherein the solution            having pH responsiveness to carbon dioxide is in liquid            communication with the liquid sample through a selectively            permeable membrane, and        -   (iii) a fluorescent species immobilized on a substrate            intermediate the tip member and the window, wherein the            fluorescent species immobilized on the substrate is in            liquid communication with the solution having pH            responsiveness to carbon dioxide during the measurement, and            wherein the window physically isolates the probe member from            the fluorescent species immobilized on the substrate.

In one embodiment, the light source is a light-emitting diode.

In one embodiment, the first and second detectors are photodiodes.

In one embodiment, the excitation lightguide, the first emissionlightguide, and the second emission lightguide are optical fibers.

In one embodiment, the housing comprises a tapered tube terminating withthe window.

In one embodiment, the selectively permeable membrane is permeable tocarbon dioxide.

In one embodiment, the tip member comprises a spike for puncturing asealed vessel.

The fluorescent species useful in the method include those noted abovein regard to the method for measuring pH and described in further detailbelow. In one embodiment, the fluorescent species comprises aseminaphthofluorescein/human serum albumin conjugate.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A is a schematic illustration of a representative system of theinvention for measuring pH;

FIG. 1B is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH;

FIG. 2 is a schematic illustration of a representative housing forexcitation and emission light guides useful in the system of theinvention;

FIG. 3 illustrates the relationship between the excitation/emissionoptical fiber housing and the sealed vessel port;

FIGS. 4A-4C illustrate a representative port assembly for introducing asubstrate-immobilized fluorescent species into a sealed vessel, FIG. 4Aillustrates the assembled port, FIG. 4B is an exploded view of the portassembly; and FIG. 4C is a plan view of tip;

FIGS. 5A and 5B illustrate representative sealed vessels incorporatingthe substrate-immobilized fluorescent species, FIG. 5A shows a sealedvessel in which substrate was introduced through process of puncture andreseal, FIG. 5B shows a sealed vessel incorporating substrate duringvessel manufacture;

FIG. 6 is a representative port assembly useful in the manufacture of asealed vessel;

FIGS. 7A-E illustrate the structures of representativeseminaphthofluorescein compounds useful in the method and system of theinvention;

FIG. 8 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (SNAFL-1) useful in the method andsystem of the invention;

FIG. 9 illustrates the emission spectra as a function of pH of arepresentative fluorescent species (EBIO-3) useful in the method andsystem of the invention;

FIG. 10 is a schematic illustration of the preparation of arepresentative fluorophore-protein (EBIO-3/HSA) conjugate useful in themethod and system of the invention;

FIG. 11 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA) useful in themethod and system of the invention;

FIG. 12 illustrates the emission spectra as a function of pH of arepresentative fluorophore-protein conjugate (EBIO-3/HSA) useful in themethod and system of the invention;

FIG. 13 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (Oxyphen);

FIG. 14 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (SNAFL-1/HSA) as afunction of pH (nitrocellulose);

FIG. 15 illustrates the emission spectra of a representativesubstrate-immobilized fluorophore-protein conjugate (EBIO-3/HSA) as afunction of pH (nitrocellulose);

FIG. 16 illustrates the data used in the method of the invention formeasuring pH;

FIG. 17 illustrates the results of the method of the invention forplatelet rich plasma;

FIG. 18 illustrates the correlation of pH results for platelet richplasma obtained by the method and system of the invention;

FIG. 19 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention;

FIG. 20 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel;

FIG. 21 illustrates the effect of probe position on fluorescentintensity in measuring pH in accordance with the invention; and

FIG. 22 illustrates the effect of membrane pore size on fluorescentintensity in measuring pH in accordance with the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for measuring pH and a systemfor measuring pH. The method and system are suited to measure the pH ofa specimen contained in a sealed container.

In one aspect of the invention, a method for measuring pH is provided.In the method, pH is determined by comparing fluorescent emissionintensities from a single fluorescent species having pH-dependentfluorescent emission. The fluorescent species having pH-dependentfluorescent emission has a first emission intensity at a firstwavelength and a second emission intensity at a second wavelength, thefirst and second emission intensities being characteristic of pH in theenvironment of the fluorescent species. The ratio of the first andsecond emission intensities provides pH measurement. Calibration of thefirst and second emission intensities provides an intensity-basedreference (ratio information) that is used to determine the pH of theenvironment of the fluorescent species.

The method of the invention is a fluorescent wavelength-ratiometricmethod. As used herein, the term “fluorescent wavelength-ratiometric”refers to the method by which the first and second fluorescent emissionintensities measured at first and second emission wavelengths,respectively, are ratioed to provide pH information.

In the method, the fluorescent species having pH-dependent fluorescentemission is immobilized on a substrate in contact with the sample suchthat the fluorescent species is in contact with the sample. Theimmobilized fluorescent species in contact with the sample is located inthe sample such that the fluorescent species can be interrogated. Thefluorescence measurement is made by irradiating the fluorescent speciesat a wavelength sufficient to elicit fluorescent emission, which is thenmeasured. Because of the pH-dependent nature of the fluorescent species'emission profile (i.e., first and second fluorescent emissionintensities measured at first and second emission wavelengths,respectively) the measurement of the fluorescent emission profile yieldsthe pH of the fluorescent species' environment (i.e., sample pH).

In one embodiment of the method of the invention, the sample for whichthe pH is to be determined is contained in a sealed vessel. This methodis suitable for measuring pH of blood and blood products sealed in aconventional blood storage vessel.

In another embodiment, the sample for which the pH is to be determinedis contained in an open vessel. As used herein, the term “open vessel”refers to a vessel that is not sealed. This method is suitable wherecontamination of the sample being measured is to be avoided. In thismethod, the probe is cleaned and/or sterilized and is used once anddiscarded. This method is suitable for measuring the pH of materialsused in food, pharmaceutical, or biological research where the vesselcontaining the material is not sealed (i.e., open). Such a “lab-use”system includes a tip (see description below) placed onto the probe. ThepH measurement is made by immersing the tip into the sample andmeasuring pH. The tip is removed from the sample, removed from theprobe, and discarded.

In the method for measuring the pH of a contained sample, thesubstrate-immobilized fluorescent species is introduced into the vesseleither before or after the sample is placed in the vessel. As usedherein, the term “sealed vessel” refers to a vessel that prevents itscontents from exposure to the environment exterior to the vessel. Thesealed vessel prevents the contents of the vessel from contact from, forexample, liquids and gases outside of the vessel. The sealed vessel alsoprevents the contents of the vessel from escaping the vessel.

The vessel can be manufactured to include the substrate-immobilizedfluorescent species as a component of the vessel. In such an embodiment,the substrate-immobilized fluorescent species is incorporated into thevessel during manufacture to provide a vessel into which a sample can belater introduced and its pH measured. The manufacture of a vesselincorporating the substrate-immobilized fluorescent species is describedin Example 1.

Alternatively, the substrate-immobilized fluorescent species can beintroduced into the vessel after the sample has been introduced into thevessel. In such an embodiment, the substrate-immobilized fluorescentspecies is introduced into the vessel by a process in which the vesselis first punctured (or spiked) to introduce the substrate-immobilizedfluorescent species and then resealed to provide a sealed vesselincluding the sample now in contact with the substrate-immobilizedfluorescent species. The process for introducing thesubstrate-immobilized fluorescent species into a sealed vessel isdescribed in Example 2.

As noted above, the vessel including the substrate-immobilizedfluorescent species in contact with the sample is sealed before, during,and after interrogation. Interrogation of the fluorescent speciesrequires excitation of the species at a wavelength sufficient to effectfluorescent emission from the species and measurement of thatfluorescent emission. In the method of the invention, interrogation isaccomplished through a window in the sealed vessel. The fluorescentspecies is excited by irradiation through the window, and emission fromthe fluorescent species is collected from the fluorescent species thoughthe window. The window is a component of the sealed vessel and allowsfor interrogation of the fluorescent species in contact with the sample.The window is sufficiently transparent at the excitation and emissionwavelengths to permit interrogation by the method. Thesubstrate-immobilized fluorescent species is positioned in proximity tothe window sufficient for interrogation: proximity sufficient toeffectively excite the fluorescent species and to effectively collectemission from the fluorescent species. It will be appreciated that forepifluorescence applications, a single window is used. However, othermethods and devices of the invention can include other optical paths,such as straight-through or right angle optical paths, where more thanone window can be used.

The method of the invention includes irradiating thesubstrate-immobilized fluorescent species, which in one embodiment iscontained along with a sample in a sealed vessel, at a wavelengthsufficient to effect emission from the fluorescent species and tomeasure that emission. Exciting light and fluorescent emission passthrough the sealed vessel's window. In one embodiment, the sealed vesselfurther includes a port for receiving a housing that holds theexcitation light guide and emission light guide. In one embodiment, theexcitation light guide includes one or more optical fibers that transmitthe excitation light from a light source to the fluorescent species. Inone embodiment, the emission light guide includes one or more opticalfibers that transmit the emission light from the fluorescent species toa light detector. The port receiving the housing is positioned inproximity to the window sufficient for interrogation: proximitysufficient to effectively excite the fluorescent species and toeffectively collect emission from the fluorescent species.

As with all optical fluorescent methods, the method of the inventionincludes a light source for exciting the fluorescent species and adetector for measuring the emission of the fluorescent species. Lightsources, wavelength selection filters, and detectors are selected basedon the absorbance and emission profiles of the fluorescent species usedin the method.

Suitable light sources provide excitation energy at a wavelength andintensity sufficient to effect fluorescent emission from the fluorescentspecies. The light source can provide relatively broad wavelength bandexcitation (e.g., ultraviolet or white light sources) or relativelynarrower wavelength band excitation (e.g., laser or light-emittingdiode). To enhance excitation efficiency and emission measurement,relatively broad wavelength band exciting light from the source can beselected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitablelight sources include tungsten lamps, halogen lamps, xenon lamps, arclamps, LEDs, hollow cathode lamps, and lasers.

Suitable detectors detect the intensity of fluorescent emission over theemission wavelength band of the fluorescent species. To enhance emissionmeasurement, fluorescent emission from the fluorescent species sourcecan be selected and narrowed through the use of diffraction gratings,monochromators, or filters to suit the fluorescent species. Suitabledetectors include photomultiplier tubes and solid state detectors, suchas photodiodes, responsive to the wavelength emission band of thefluorescent species. Other suitable detectors are photovoltaic cells,PIN diodes, and avalanche photodiodes.

Through the use of filters, all of the excitation light that reflectsoff the target is filtered out before reaching the detector. This can beachieved by using filters in both the excitation and emission opticalpaths. In certain instances, reflected excitation light (which is manyorders of magnitude more intense than the emission light) that reachesthe detector can swamp the specific signal. Generally, 10E5 (10⁵) orgreater out-of-band rejection is appropriate in each of the filter sets.Reduction of excitation light can also be achieved by using an angledwindow so that reflected light is directed away from the emissiondetector. However, such an optical path is not as effective as filtersets.

Excitation light from the source can be directed to the fluorescentspecies through the use of a light guide, such as one or more opticalfibers. Similarly, emission from the fluorescent species can be directedto the detector through the use of a light guide, such as one or moreoptical fibers.

A representative system for carrying out the method of the invention isillustrated schematically in FIG. 1A. Referring to FIG. 1A, system 100includes controller 110 that controls and operates the systemcomponents. System components include keypad 120 for inputtinginformation including system commands; display 130 for determining thestatus of the system and viewing pH determination results; barcodereader 140 for inputting information to the system including theidentification of the sample, the pH of which is to be measured by thesystem; printer 150 for printing system status and pH determinationresults; battery (or wall plug and power adapter) 160 for powering thesystem; memory device 165 for storing test results and calibration data;signal processing electronics 170 for commanding the optical platformcomponents and processing signals from the optical platform; and opticalplatform 180 including an excitation source, emission detectors, lightguides, and associated lenses and filters. Optical platform includesprobe member 185 housing one or more excitation light guides and two ormore emission light guides. FIG. 1A also illustrates sealed vessel 500including port 205 for receiving probe member 185.

FIG. 1B is a schematic illustration of an optical platform useful in thesystem of the invention for measuring pH. Referring to FIG. 1B, opticalplatform 180 includes excitation optics 280, first emission optics 380,and second emission optics 480. Excitation optics 280 include lightsource 282, collimating lens 284, filter 286, focusing lens 288, andexcitation light waveguide 290. First emission optics 380 includedetector 382, focusing lens 384, filter 386, collimating lens 388, andfirst emission light waveguide 390. Second emission optics 480 includesdetector 482, focusing lens 484, filter 486, collimating lens 488, andsecond emission light waveguide 490. Excitation light guide 290, firstemission light waveguide 390, and second emission light waveguide 490are housed in probe member 185.

The system's light source is effective in exciting the fluorescentspecies. Suitable light sources include light-emitting diodes, lasers,tungsten lamps, halogen lamps, xenon lamps, arc lamps, and hollowcathode lamps. In one embodiment, the light source is a light-emittingdiode emitting light in the range from 500 to 560 nm. A representativelight-emitting diode useful in the system of the invention is a greenultrabright Cotco 503 series LED commercially available from Marktech,Latham N.Y.

The collimating lens directs light (e.g., excitation light from thelight source or first and second emission light from the emission lightwaveguides) to the bandpass filter. Suitable collimating lenses includeBiconvex glass lenses and Plano-convex glass lenses. Representativecollimating lenses useful in the system of the invention are the TechSpec PCX lenses commercially available from Edmund Optics, Barrington,N.J. The excitation collimating lens is 12×36 (diameter by effectivefocal length in mm) and the first and second emission collimating lensesare 12×18.

The focusing lens focuses light from the bandpass filter to theexcitation light waveguide or from the bandpass filter to the detector.Suitable focusing lenses include Biconvex glass lenses and Plano-convexglass lenses. Representative focusing lenses useful in the system of theinvention are the Tech Spec PCX lenses commercially available fromEdmund Optics, Barrington, N.J. The excitation focusing lens is 12×18and the first and second emission focusing lenses are 12×15.

Filters are used in the optical platform to narrow the bandwidth oftransmitted light.

Suitable excitation filters include bandpass filters, shortpass filters,longpass filters, or a combination of short and long pass filters. Inone embodiment, the system uses a shortpass filter that passes light inthe range from about 370 nm to 540 nm. A representative excitationshortpass filter useful in the system of the invention is 540ASPcommercially available from Omega Optical, Brattleboro, Vt.

Suitable first emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 595 to 605 nm andhas a full width at half height of 10 nm. A representative firstemission bandpass filter useful in the system of the invention is600DF10 commercially available from Omega Optical, Brattleboro, Vt.

Suitable second emission filters include bandpass, shortpass, longpass,or a combination of short and longpass filters. In one embodiment, thebandpass filter passes light in the range from about 562 to 573 nm andhas a full width at half height of 10 nm. A representative secondemission bandpass filter useful in the system of the invention is568DF10 commercially available from Omega Optical, Brattleboro, Vt.

The excitation light waveguide transmits excitation light from the lightsource through the probe member to the fluorescent species. In oneembodiment, the excitation light waveguide includes one or more opticalfibers. In one embodiment, the excitation waveguide is a single opticalfiber. A representative fiber optic useful in the system of invention isRO2-534 commercially available from Edmund Optics, Barrington, N.J.

The first and second emission light waveguides transmit fluorescentemission from the fluorescent species through the probe member to thefirst and second emission detectors, respectively.

In one embodiment, the first emission light waveguide includes one ormore optical fibers. In one embodiment, the first emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thefirst emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is RO2-533commercially available from Edmund Optics, Barrington, N.J.

In one embodiment, the second emission light waveguide includes one ormore optical fibers. In one embodiment, the second emission lightwaveguide includes a plurality of optical fibers. In one embodiment, thesecond emission light waveguide includes four optical fibers. Arepresentative fiber optic useful in the system of invention is R02-533commercially available from Edmund Optics, Barrington, NJ.

Suitable optical fibers useful in the system of the invention includeglass or plastic optical fibers from 0.2 to 2 mm diameter.

The system's first and second emission detectors are effective inmeasuring the first and second fluorescent emissions from thefluorescent species. Suitable detectors include photodiodes, PIN diodes,and photomultiplier tubes. In one embodiment, the first and secondemission detectors are photodiodes responsive in the range from 400 to800 nm. Representative photodiodes useful in the system of the inventioninclude BPW34 commercially available from Vishay Intertechnology,Malvern, Pa.

A representative probe member housing excitation and emission lightguides useful in the system of the invention is illustratedschematically in FIG. 2. As shown in FIG. 2, the light guides areoptical fibers. Referring to FIG. 2, probe member 185 houses excitationlight guide 290, a plurality of first emission light guides 390, and aplurality of second emission light guides 490. In the representativeprobe member shown in FIG. 2, there are four first emission light guides390, and four second emission light guides 490. The four first emissionlight guides can be considered to be a first channel (e.g., measuringthe first fluorescent emission from the fluorescent species) and thefour second emission light guides can be considered to be a secondchannel (e.g., measuring the second fluorescent emission from thefluorescent species). In the illustrated representative probe member,the fibers from each of the two sets of fibers alternate (i.e.,alternating fibers 390 and 490) around the central fiber (290). Thisconfiguration provides for evening out of “hot spots” so that lightcollected by the first set is similar to the light collected by thesecond set.

The relationship between the probe member housing theexcitation/emission light guides and the sealed vessel port isillustrated schematically in FIG. 3. Referring to

FIG. 3, probe member 185 is received by port 205. Port 205 includeswindow 210, which is transparent to excitation and emission wavelengthsused in the fluorescent measurement. Excitation light emanating fromlight guide 290 passes through window 210 and interrogates substrate 220on which the fluorescent species is immobilized and which, in theoperation of the method of the invention, is in contact with the samplecontained in sealed vessel 200. Irradiation of substrate 220 results inexcitation of the substrate-immobilized fluorescent species andfluorescent emission from the fluorescent species. Emission from thefluorescent species is received by and transmitted through light guides390 and 490 to detectors 382 and 482, respectively (see FIG. 1B). Asnoted above, the fluorescent species' first emission intensity and thesecond emission intensity will depend on the pH of the sample.

A representative port assembly for introducing the substrate-immobilizedfluorescent species into a sealed vessel is illustrated in FIGS. 4A and4B. FIG. 4A illustrates the assembled port and FIG. 4B is an explodedview of the port assembly. Referring to FIGS. 4A and 4B, port assembly202 includes port 205 and tip 215. Port 205 is a cylinder terminatingwith window 210 and having opening 212 for receiving probe member 185(not shown). In one embodiment, port 205 tapers from opening 212 towindow 210 such that the depth of insertion of probe member 185 intoport 205 is predetermined by the probe's diameter. In one embodiment,the depth of travel of 185 in assembly 202 is limited by a ledge (notshown). In one embodiment, the optimal distance between probe andmembrane was determined to be 2 mm or less. FIG. 21 illustrates thefluorescence intensity measured as a function of distance between theprobe and membrane. When inserted in the port, the face of probe member185 and window 210 are substantially parallel. Port 205 and tip 215 areadapted such that the port and tip are reversibly connectable. In oneembodiment, port 205 includes annular inset 214 and tip 215 includesopening 216 defined by annular lip 218 for receiving inset 214. In thisembodiment, inset 214 has a diameter less than opening 216. It will beappreciated that the connecting relationship between the port and tipcan be reversed (i.e., port having annular lip for receiving tip havinginset). Lip 218 defines bed 222 for receiving substrate 220, which issecured in port assembly 202 when port 205 is connected to tip 215. Tip215 includes aperture 224 in bed 222. Aperture 224 provides for contactof substrate 220 with a liquid sample contained in a sealed vessel intowhich port assembly is introduced. Tip 215 terminates with apex 226 thatfacilitates the introduction of port assembly 202 into a sealed vesselby puncture. FIG. 4C is a plan view of tip 215 illustrating bed 222 andaperture 224. In one embodiment, the assembly is made from Lexan HPS11125 available from GE Polymerland, Pittsfield, Mass.

Representative sealed vessels incorporating the substrate-immobilizedfluorescent species are illustrated in FIGS. 5A and 5B. FIG. 5Aillustrates a sealed vessel into which a port assembly has been insertedby puncture. FIG. 5B illustrates a sealed vessel manufactured to includea port assembly.

Referring to FIG. 5A, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 202 (including port 205, membrane 220, and tip215) resides in vessel port 510A after insertion. Vessel 500 remainssealed after insertion of port assembly 202. Vessel port 510A seals toport 205.

Referring to FIG. 5B, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 232 (including port 205, membrane 220, and tip215) resides in vessel port 510A after vessel manufacture. A process formanufacturing a representative sealed vessel incorporating a portassembly is described in Example 1.

A representative port assembly useful for incorporation into a sealedvessel during manufacture is illustrated in FIG. 6. The port assemblyuseful for incorporation during vessel manufacture is substantially thesame as the port assembly useful for introduction into a sealed vesselillustrated in FIG. 4, except that the assembly useful in vesselmanufacture need not include, and preferably does not include, a featurefor puncturing the vessel. Referring to FIG. 6, port assembly 232includes port 205 and tip 235. Port 205 is a cylinder terminating withwindow 210 and having opening 212 for receiving probe member 185 (notshown). In one embodiment, port 205 tapers from opening 212 to window210 such that the depth of insertion of probe member 185 into port 205is predetermined by the probe's diameter. When inserted in the port, theface of probe member 185 and window 210 are substantially parallel. Port205 and tip 235 are adapted such that the port and tip are reversiblyconnectable. In one embodiment, port 205 includes annular inset 214 andtip 235 includes opening 216 defined by annular lip 218 for receivinginset 214. In this embodiment, inset 214 has a diameter less thanopening 216. It will be appreciated that the connecting relationshipbetween the port and tip can be reversed (i.e., port having annular lipfor receiving tip having inset). Lip 218 defines bed 222 for receivingsubstrate 220, which is secured in port assembly 202 when port 205 isconnected to tip 235. Tip 235 includes aperture 224 in bed 222. Aperture224 provides for contact of substrate 220 with a liquid sample containedin the sealed vessel.

Fluorescent species having pH-dependent emission. The method and systemof the invention for measuring pH uses a fluorescent species havingpH-dependent fluorescent emission. The fluorescent species has a firstemission intensity at a first wavelength and a second emission intensityat a second wavelength, the first and second emission intensities beingcharacteristic of pH in the environment of the fluorescent species. Theratio of the first and second emission intensities provides pHmeasurement. It is appreciated that fluorescent emission occurs as awavelength band having a band maximum that is referred to herein as theemission wavelength.

In one embodiment, the separation between the first wavelength and thesecond wavelength is at least about 40 nm. In one embodiment, theseparation between the first wavelength and the second wavelength is atleast about 30 nm. In one embodiment, the separation between the firstwavelength and the second wavelength is at least about 20 nm. Using 10nm HBW filters, the separation is at least about 30 nm. Preferably, thesystem of the invention achieve fluorescence signal separation byremoving any emission band overlap by 10E5 or more. The method andsystem of the invention for measuring pH are not limited to anyparticular fluorescent species, nor any particular pH range. The methodand system of the invention is operable with any fluorescent specieshaving pH-dependent properties that can be excited and its emissionmeasured. The range of pH measurable by the method and system of theinvention can be selected and is determined by the pH-dependentproperties of the fluorescent species.

In addition to their pH-dependent properties noted above, suitablefluorescent species include those that can be substantially irreversiblyimmobilized on a substrate. The fluorescent species can be covalentlycoupled to the substrate or non-covalently associated with thesubstrate.

Suitable pH-dependent fluorescent species include those known in theart. Representative fluorescent species having suitable pH-dependentproperties include fluorescein derivatives including naphthofluoresceincompounds, seminaphthofluorescein compounds (e.g., SNAFL compounds), andseminaphthorhodafluor compounds (e.g., SNARF compounds). These compoundshave advantages associated with their long wavelength emission, which isless susceptible to potential interfering light absorbing substances inblood. These compounds also have relatively long wavelength absorbancemaking them particularly suitable for excitation by commerciallyavailable LED light sources. Another compound having suitable pHdependent behavior is HPTS, 8-hydroxy-1,3,6-pyrenetrisulfonic acid.Although the compound has desired ratiometric pH properties, excitationis optimal at short wavelength (403 nm) where strong LED light sourcesare not commercially available. Representative SNAFL and SNARF compoundsuseful in the method and system of the invention are described in U.S.Pat. No. 4,945,171. Molecular Probes (now Invitrogen, Eugene, Oreg.)sells CNF, SNAFL, SNARF fluors with conjugatable carboxylic acid linkergroups, see, for example, Molecular Probes Handbook (Ninth Edition) byR. P. Haugland, Chapter 21 “pH indicators” pages 829-847. EpochBiosciences (now Nanogen, Bothell, Wash.) sells EBIO-3 with a propanoicacid linker. Whitaker et al. (Anal. Biochem. (1991) 194, 330-344) showedthe synthesis of a number of SNAFL compounds. Wolfbeis et al. (MikrochimActa (1992) 108, 133-141) described the use of CNF and aminocelluloseconjugates. The earliest reference to the SNAFL family of compounds isWhitaker et al. (1988) Biophys. J. 53, 197a. A related dye in the CNFfamily is VITABLUE, a sulfonenaphthofluorescein derivative (Lee et al(1989) Cytometry 10, 151-164) having a pKa of 7.56. A CNF analog withbromine substituents at each carbon adjacent to a phenol (pKa 7.45) hasa pKa that is 0.54 pKa units lower than their measured pKa for CNF (pKa7.99). Lee et al. note that “true” pKa values are difficult to determinefor these compounds. A method for pKa determination is described inExample 3. SNAFL-1 (literature pKa˜7.8) free acid had a pKa of 7.6 inthat fluorescence-based assay.

The structures of seminaphthofluorescein compounds (SNAFL-1 and EBIO-3)useful in the method and system of the invention are illustrated below.

The numbering scheme describes position of attachment of linkermolecules. These compounds have carboxylate linking groups suitable forconjugation to carrier proteins, as described below. For conjugation,the reactive N-hydroxysucinimide (NHS) ester of SNAFL-1 (commerciallyavailable from Molecule Probes, Inc., Eugene, Oreg.) can be used.Conjugation to lysine residues in human serum albumin (HSA) gave desired

SNAFL/HSA conjugates. Carbodiimide activation of EBIO-3 gave a reactiveintermediate that was efficiently conjugated to human serum albumin.

Representative naphthofluorescein and seminaphthofluorescein compoundsuseful in the method and system of the invention are illustrated in FIG.7.

The SNAFL compounds are commercially available from Molecular Probes,Inc., Eugene, Oreg. The SNAFL compounds can be readily synthesizedaccording to general procedures that have been published (see, forexample, U.S. Pat. No. 4,945,171).

The preparation of a representative 2-chloro substituted SNAFL compoundis shown below.

The compound can be prepared by condensation of 1,6-dihydroxynaphthalenewith the diacid substituted 4-acylresorcinol in the presence of adehydrating acid or Lewis acid catalyst, such as Zinc chloride.

The preparation of SNAFL compounds having propionic acid linkers isdescribed in U.S. patent application Ser. No. 11/022,039, incorporatedherein by reference in its entirety. A representative SNAFL compoundshaving a propionic acid linker, EBIO-3, is commercially available fromNanogen, Bothell Wash.

The emission spectra as a function of pH of representative fluorescentspecies (i.e., SNAFL-1 and EBIO-1) useful in the method and system ofthe invention are illustrated in FIGS. 8 and 9, respectively. FIG. 8illustrates the emission spectra of SNAFL-1 in 50 mM potassium phosphatebuffer as a function of pH (pH 6.0 to 10.0) (excitation at 540 nm).Referring to FIG. 8, the response at pH 6-7 is relatively poor(pKa=7.6). FIG. 9 illustrates the emission spectra of EBIO-3 in 50 mMpotassium phosphate buffer as a function of pH (pH 6.0 to 10.0)(excitation at 545 nm). Referring to FIG. 9, the response at pH 6-7 isrelatively good (pKa=6.6). Spectral properties and pKa data for theSNAFL analogs illustrated in FIGS. 7A-7E are summarized in Table 1.

TABLE 1 pH-Sensitive absorbance and emission of SNAFL analogs. EmissionAbsorbance Absorbance Emission λmax Compound λmax (acid) λmax (base)λiso (base) pKa SNAFL-1 482, 510 nm 540 nm 585 nm 620 nm 7.6 SNAFL-2485, 514 547 590 630 7.6 EBIO-1 496, 519 545 560 620 6.5 EBIO-2 506, 538572 590 645 7.8 EBIO-3 480, 509 534 560 610 6.6

Referring to Table 1, absorbance and emission spectra were obtained at10 μM SNFL analog. Absorbance was measured at pH 6, 8, and 10: acid (pH6) gave two bands of similar absorbance; pH 10 gave a single λmax(base). The emission spectra were determined by excitation at theabsorbance λmax (base). The wavelength where emission spectra crossed isreported as λiso. The emission λmax was measured at pH 10. pKa wasdetermined from fluorescence emission spectra. EBIO-1 and EBIO-3 weremore sensitive to changes at pH˜6.5. The other analogs were moresensitive at pH˜8.

Fluorescent species conjugates for substrate immobilization. For use inthe method and system of the invention, the fluorescent species isimmobilized on a substrate such that the fluorescent species is incontact with the sample, the pH of which is to be measured. Thefluorescent species can be immobilized on the substrate through the useof a material (e.g., macromolecular spacer material) having a strongassociative interaction with the substrate. The spacer material allowscovalent conjugation of the fluorescent species and provides largesurface area needed for efficient non-covalent immobilization to thesubstrate surface. In one embodiment, the spacer material is human serumalbumin (HSA) having ˜44 lysine residues available for covalentconjugation. HSA's densely charged molecular structure has a passivatingeffect when adsorbed to biomaterials. Other advantages include reducedfluorescence quenching, uniform environment for the conjugatedfluorophore, and availability in recombinant form (from yeast) so thereis no chance of infection (as with HSA from donors). HSA conjugates areeasily purified by ultrafiltration methods and form stable solutionsthat are easily characterized by absorbance and fluorescence assays todetermine the number of fluorophores per protein.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a protein or protein fragment. Suitableproteins include those that can be substantially irreversiblyimmobilized on the substrate. The protein can be covalently coupled tothe substrate or non-covalently associated with the substrate. Suitableproteins include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the protein.

Suitable proteins include human serum albumin (HSA), bovine serumalbumin (BSA), vonWillebrand's factor, kininogen, fibrinogen, andhemoglobin (no iron). Suitable proteins include proteins havingavailable lysine residues (for conjugation to the fluorophore) andmolecular weight sufficient to allow for immobilization efficiency tothe blot membrane. Other functional groups in the protein (likecysteine) could presumably be used for covalent bonding to suitablyreactive solid supports.

In one embodiment, the fluorescent species is immobilized on thesubstrate through the use of a polysaccharide. Suitable polysaccharidesinclude those that can be substantially irreversibly immobilized on thesubstrate. The polysaccharide can be covalently coupled to the substrateor non-covalently associated with the substrate. Suitablepolysaccharides include proteins to which the fluorescent species can besubstantially irreversibly immobilized. The fluorescent species can becovalently or non-covalently associated with the polysaccharide.

Suitable polysaccharides include dextrans, aminodextrans, heparin, andlectins.

In another embodiment, the fluorescent species is immobilized on thesubstrate through the use of dendrimeric structures. Suitabledendrimeric structures include those that can be substantiallyirreversibly immobilized on the substrate. The dendrimeric structurescan be covalently coupled to the substrate or non-covalently associatedwith the substrate. PAMAM dendrimers are commercially available as areother structural types and sizes.

In one embodiment, the fluorescent species is covalently coupled to aprotein to provide a fluorophore-protein conjugate that can beimmobilized on a substrate. In one embodiment, thefluorophore-polysaccharide conjugate is non-covalently associated withthe substrate.

In one embodiment, a fluorophore-protein conjugate is immobilized on asubstrate. In one embodiment, the fluorescent species is aseminaphthofluorescein and the protein is human serum albumin. In oneembodiment, the seminaphthofluorescein is SNAFL-1. The preparation ofSNAFL-1/HSA conjugates is described in Example 4. The fluorescentproperties of SNAFL-1/HSA conjugates are described in Example 5. In oneembodiment, the seminaphthofluorescein is EBIO-3. The preparation ofEBIO-3/HSA conjugates is described in Example 6. A schematicillustration of the coupling of EBIO-3 to HSA is illustrated in FIG. 10.The fluorescent properties of EBIO-3/HSA conjugates are described inExample 7.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (SNAFL-1/HSA, 1.6fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 11.

The fluorescent emission spectra as a function of pH (6.0 to 10.0) of arepresentative fluorophore-protein conjugate (EBIO-3/HSA, 1.92fluorophores per HSA) useful in the method and system of the inventionare illustrated in FIG. 12.

For the fluorophore-protein conjugate, the optimum fluorophore loadingwill vary depending on the particular fluorophore.

For SNAFL-1/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 SNAFL-1/HSA. Low signal at 0.01 and fluorescentquenching at 40 fluorophores/HSA. In one embodiment, the SNAFL-1conjugate includes about 2 SNAFL-1/HSA.

For EBIO-3/HSA conjugates the fluorophore loading can vary from about0.01 to about 40 EBIO-3/HSA. In one embodiment, the EBIO-3 conjugateincludes about 2 EBIO-3/HSA.

Substrates for fluorescent species immobilization. In the method andsystem of the invention, the fluorescent species is immobilized on asubstrate. As noted above, the fluorescent species can be directlyimmobilized on the substrate covalently or by non-covalent associationor, alternatively, through the use of a material (e.g.,fluorophore-protein conjugate) that can be immobilized on the substratecovalently or by non-covalent association.

Suitable substrates substantially irreversible immobilized thefluorescent species. In the method of the invention, suitable substratesalso do not inhibit the contact of the liquid sample with thefluorescent species and do not impair or alter the pH measurement.

Representative substrates include membranes, such as microporousmembranes made of nitrocellulose, mixed esters of nitrocellulose andcellulose acetate, polyethylene terephthalate, polycarbonate,polyvinylidene fluoride and polyimide. Such materials are availablecommercially from Whatman S&S, Florham Park, N.J. and Millipore,Billerica Mass. Suitable membranes include membranes in which themicroporous structure is created by ion beam penetration such asmembranes commercially available from Oxyphen Gmbh, Dresden, Germanyunder the designation OXYPHEN. Charged nylon surfaces (Nytran) can alsobe used. Suitable membranes include plastic membranes in which themicroporous structure is made by injection molding the micropores intothe plastic such as the processes used by Åmic, Stockholm, Sweden.Emission intensity of SNAFL-1/HSA at pH 7 immobilized on various poresize mixed ester nitrocellulose cellulose acetate membranes is shown inFIG. 22.

Immobilization of representative fluorophore protein conjugates onmembranes is described in Examples 8 and 10. Example 8 describes theimmobilization of SNAFL-1/HSA conjugates. Example 9 describes thefluorescent properties of immobilized SNAFL-1/HSA conjugates. Example 10describes the immobilization of EBIO-3/HSA conjugates. Example 11describes the fluorescent properties of immobilized EBIO-3/HSAconjugates.

The emission spectra of a representative fluorophore-protein conjugate(SNAFL-1/HSA, 3.6:1) immobilized on Oxyphen and nitrocellulose as afunction of pH (pH response), as measured by the microwell assaydescribed in Example 9, are illustrated in FIGS. 13 and 14,respectively.

The emission spectra of a representative fluorophore-protein conjugate(EBIO-3/HSA, 2.0:1) immobilized on nitrocellulose, as described inExample 8, as a function of pH (6.0, 6.5, 7.0, 7.5, 8.0, and 10.0), asmeasured by the telescoping tube insert assay described in Example 11,are illustrated in FIG. 15. The large spread of emissions at 600 nm forthe pH 6 to 8 range indicates good fluorescence verses pH response.

Ratiometric pH Method and System. The method of the invention is afluorescent wavelength-ratiometric method. In the method, the first andsecond fluorescent emission intensities of the fluorescent speciesmeasured at first and second emission wavelengths, respectively, areratioed to provide pH information. The first emission wavelength varieswith pH while the second emission wavelength is constant with pH andgives an internal control for the fluorescent intensity. In oneembodiment, a lookup table is used to lookup a combination of themeasured ratio, first and second emission wavelength and determines itscorresponding pH. In one embodiment, a mathematical function of theratio, first and second emission wavelength is used to calculate theresulting pH.

FIG. 16 illustrates the data used in the method of the invention formeasuring pH. The emission spectra of a representativefluorophore-protein conjugate (EBIO-3/HSA, 2:1) immobilized onnitrocellulose at pH 7.0 is shown as measured by the telescoping tubinginsert assay. In this setup, the excitation bandpass filter was unableto completely remove the excitation light in the emission region asillustrated by the background signal measured on a blank nitrocellulosedisc. The full spectrum corrected for the background was multiplied bythe transmittance of the first and second hypothetical filters at eachwavelength and the area under the resultant curve was calculated to givea signal for the first and second wavelength. The center wavelengths andbandwidths of hypothetical filters were chosen such that the ratiometricproperties of the conjugate had the strongest relationship to the pH inthe region of interest.

FIG. 17 illustrates the results of the method of the invention forphosphate buffered saline (PBS), platelet poor plasma (PPP), andplatelet rich plasma (PRP) as measured by the telescoping tubing insertassay described in Example 11. The three curves represent the best fitrelationship between the measured pH and ratios for the three differentliquids.

FIG. 18 illustrates the correlation of pH results for three differentplasma samples obtained by the method and system of the invention asmeasured by the injection molded insert PVC tube assay described inExample 11. The relationship between the fluorescent signal and the pHhas an accuracy of about 0.1 pH units.

FIG. 19 illustrates stability of a representative substrate-immobilizedfluorophore conjugate of the invention (EBIO-3/HSA, 2:1) on mixed esternitrocellulose and cellulose acetate prepared by the soaking method andas measured by the leaching assay described in Example 10. The low levelof leaching is far below the toxic level for any compound.

Carbon dioxide measurement. In another aspect, the present inventionprovides a device and method for measuring carbon dioxide concentrationin a liquid sample. The carbon dioxide measuring method utilizes the pHmeasuring method and system described above. In the carbon dioxidemeasuring method and device, a substrate-immobilized fluorescent speciesas described above is in contact with a solution, the pH of which isresponsive to carbon dioxide level. In addition to being in contact withthe substrate-immobilized fluorescent species, the solution having pHresponsive to carbon dioxide level is in contact with a liquid samplefor which the level of carbon dioxide is to be measured. The solutionhaving pH responsive to carbon dioxide level is isolated from the liquidsample for which the level of carbon dioxide is to be measured by aselectively permeable membrane. The membrane is permeable to gases(e.g., carbon dioxide) and impermeable to other materials (e.g.,liquids). Using the method of measuring pH described above, the pH ofthe solution responsive to carbon dioxide concentration in contact withthe substrate-immobilized fluorescent species is measured and correlatedwith the carbon dioxide level of the sample in contact with thatsolution.

The solution having pH response to carbon dioxide level is an aqueoussolution that includes an agent that is reactive toward carbon dioxideand changes the pH of the solution in response to carbon dioxideconcentration. Suitable agents that are reactive toward carbon dioxideand change the pH of the solution in which they are dissolved includebicarbonates, such as sodium bicarbonate.

The selectively permeable membrane isolates the solution having pHresponsive to carbon dioxide level from the liquid sample containingcarbon dioxide. The membrane is permeable to carbon dioxide andimpermeable to liquids and other solutes. In the method, carbon dioxidefrom the liquid sample passes from the liquid sample through themembrane and into the aqueous solution thereby reacting with the carbondioxide reactive agent to alter the pH of the aqueous solution. Suitableselectively permeable membranes include membranes made from silicone andPTFE.

FIG. 20 illustrates a representative device of the invention formeasuring carbon dioxide in a sealed vessel. Referring to FIG. 20,device 600 includes port assembly 610 including port 620 and tip 630.Port 620 is a cylinder terminating with window 622 and having opening624 for receiving probe member 185. When inserted in the port, the faceof probe member 185 and window 622 are substantially parallel. Port 620and tip 630 are adapted such that the port and tip are reversiblyconnectable. Substrate 640 including immobilized fluorescent species issecured within port 620 and tip 630. Tip 630 includes a chamber 645 forreceiving a solution having pH responsiveness to carbon dioxide. Chamber645 is defined by window 622, tip 630, and selectively permeablemembrane 650. Chamber 645 includes substrate 640, which is interrogatedby probe member 185.

A device for measuring carbon dioxide was assembled as described abovewith the membrane containing immobilized EBIO-3/rHSA conjugate (rHSA isrecombinant

HSA). A layer of PARAFILM M, a blend of olefin-type materials, was addedunder the membrane towards the tip. The membrane was hydrated with 5 ulof 35 mM carbonate buffer (pH 7.4), which was sealed within the assemblyby the PARAFILM M and remained hydrated throughout the assay. Theassembly was subjected to 100% carbon dioxide gas by connection to thegas source with tubing and a “Y” adapter to bleed off the pressure. Theassembly was subjected to the carbon dioxide for an allotted period oftime, allowed to acclimate to ambient air conditions, and repeated. Thefluorescence was measured at each stage at 568 nm and 600 nm after beingexcited at 525 nm. The results are summarized below in Table 2 andreflect changes in fluorescence due to the change in carbon dioxideconcentration demonstrating that the fluorometric ratio method of theinvention can also be used to calculate carbon dioxide concentration.The PVC storage bags that are used for platelet storage are somewhat gaspermeable, and carbon dioxide is directly related to the measurement ofpH.

TABLE 2 Carbon dioxide sensing results. EM at EM at Ratio EnvironmentalConditions 568 nm 600 nm (600/568) 15 min. at Ambient CO₂ 753 2184 2.9 5 min. at 100% CO2 1179 2234 1.894 15 min. at Ambient CO2 833 21752.611  8 min. at 100% CO2 1161 1930 1.662 60 min. at Ambient CO2 7652184 2.854

The present invention provides a fluorescence-based pH indicator thatcan be easily inserted into the sampling ports of designed blood storagebags and interrogated using a fiber optic-based LED light source andphotodiode measurement system. This solid state system uses a“ratiometric” calibration method that accounts for variability influorescent signal strength due to interfering substances in blood thatmay interfere with the amount of excitation light that hits theindicator dye. The ratio of fluorescence intensities are measured at twowavelengths, one to detect the acid (protonated) isomer of the dye andone to detect the base (deprotonated) isomer.

To develop an accurate pH detector for platelet rich plasma, compoundshaving pKa of ˜6.6 are suitable, for example, 2-chloro substitution ofSNAFL compound lowers the pKa of the phenol from 7.6 to ˜6.6. Conjugatesof these compounds can be immobilized to various solid supports toprovide sensing pH membranes.

The present invention provides an inexpensive, easy to manufacture pHsensing membrane that gives accurate measurement of pH in plateletstorage bags at pH 6.5-7.5. In one embodiment, the invention uses aprotein conjugate (human serum albumin) of a 2-chloro substitutedratiometric fluorescent compound. The fluorophore:HSA ratio wasoptimized for performance when immobilized to a nitrocellulose blotmembrane. After drying on the membrane, the fluorophore:HSA conjugatehas very low leaching rates. Discs of this material are easily assembledinto holders for insertion into the sampling ports of platelet storagebags. The fluorescent membrane materials showed good pH response using agreen LED based fluorometer. In the method, two emission wavelengths forratiometric pH detection are measured with properly filtered photodiodeswith an accuracy of ˜0.1 units at the desired low pH threshold of 6.5.

Fluorescent probe molecules can be designed to be sensitive to a varietyof environments. The method and system of the invention describes theuse of pH-sensitive fluorophores. However, other environments can beinterrogated by the method and system of the invention modified toinclude environment-sensitive fluorophores other than pH-sensitivefluorophores. A variety of fluorescent probes that change fluorescentproperties as the molecular environment changes are commerciallyavailable. See, for example, Molecular Probes Handbook (9th Edition) byR. P. Haugland. Probes can be linked to albumins or other proteins andused to prepare substrates for interrogation as described in herein orusing other fluorescent-based methods. Examples of environment-sensitivefluorophores, systems, and methods include the following.

Nucleic acid detection: nucleic acid binding dyes change fluorescentproperties in the presence of DNA or RNA.

Enzyme substrates: proteins or peptides can be labeled with fluorescentdyes and fluorescent quenching molecules such that a fluorescent signalis generated in the presence of particular enzymes such as proteases(FRET detection).

Probes for lipids: lipophilic dyes can change fluorescent properties inthe presence of cell membranes or other lipid rich analytes.

Probes for oxygen: in addition to pH detection and carbon dioxidedetection, certain fluorescent molecules are sensitive to changes inoxygen concentration, for example, tris(2,2′-bipyridiyl)ruthenium(II)dichloride (RTDP).

Indicators for metal ions: fluorescent dyes that bind metals can changefluorescent properties upon binding calcium, magnesium, zinc, sodium,potassium, among other.

Glucose detection: certain lectins such as ConA bind glucose, andsuitably labeled lectins can be prepared as probes for glucose.

The following examples are provided for the purposes of illustrating,not limiting, the invention.

EXAMPLES Example 1 The Manufacture of a Vessel Incorporating aRepresentative Substrate-Immobilized Fluorescent Species

Referring to FIG. 5B, sealed vessel 500 is manufactured from PVC. PVCmaterial is compounded with a number of additives, for example,plasticizers, stabilizers, and lubricants. The formulation is used formaking bags and tubes. The compounded PVC is extruded through a die forconverting the plasticized material into sheet form. The extruded sheet,after slitting, is cut into the desired size and sent to the weldingsection. The donor and transfer tubings are made by extrusion of similarPVC compounds. The tubes are then cut to the appropriate length and sentto the welding section. The components, such as transfusion ports,needle covers, and clamp, are produced by injection molding. Thecomponents are ultrasonically cleaned and dried in a drying oven.

Welding. The blood bags are fabricated by a high frequency weldingtechnique. Sized PVC sheets are placed between electrodes and highfrequency at high voltage is applied. PVC gets heated very rapidly andsealing takes place between electrodes.

Transfusion ports and donor and transfer tubing are kept in theappropriate position with the bag and welded to form an integral part ofthe blood bag system. For the manufacture of a vessel incorporating arepresentative substrate-immobilized fluorescent species (FIG. 5B), anopen tube is welded to provide port 510A. The tube can be made ofcolored PVC to provide light protection for the immobilized fluorescentspecies. Welded bags are trimmed. The port assembly 232 (FIG. 6) ismanufactured from injection molded Lexan parts (205 and 235) and a 3.53mm ( 9/64 inch) diameter nitrocellulose disc with immobilizedfluorescent species (220). The port assembly is held together byfriction fit or can be glued in place. The port assembly is inserted inthe open tube of port 510A. The port assembly is held in the port byfriction fit or can be glued in place. The assembled bag and portassembly is sterilized and labeled for ultimate storage of plateletconcentrates.

Example 2 The Incorporation of a Representative Substrate-ImmobilizedFluorescent Species into a Sealed Vessel

Referring to FIG. 5A, sealed vessel 500 includes a plurality of vesselports 510. Port assembly 202 resides in vessel port 510A afterinsertion. The port assembly 202 (FIGS. 4A-4C) is manufactured frominjection molded Lexan parts (205 and 215) and a 3.53 mm ( 9/64 inch)diameter nitrocellulose disc with immobilized fluorescent species (220).The port assembly is held together by friction fit or can be glued inplace.

The port assembly is inserted through the septum seal inside port 510Aby puncturing the seal with the spiked tip. Alternatively, the seal canbe pre-punctured with a separate spike tool. The insertion of the portassembly can be performed on either empty or platelet filled bags, butin either case, aseptic methods should be used to avoid possiblecontamination of the bag contents. The port assembly is held in the portby friction fit or can be glued in place. Vessel 500 remains sealed(leakproof) after insertion of port assembly 202 in port 510A.

Example 3 Fluorescence and pH Properties of Representative SNAFLAnalogs: pKa Determination

Instrumentation. Fluorescence versus pH of various SNAFL free acids werecompared using an Ocean Optics USB2000 fiber optic spectrometer and atungsten halogen light source (part number HL-2000 FHSA). The lightsource was equipped with a linear variable filter that allowed thewavelength and shape of the excitation beam to be adjusted. Theexcitation wavelength was adjusted by using a blank cuvette to theabsorbance max of the fluorophore (see Table 1). A cuvette holder (partnumber CUV-FL-DA) was directly attached to the light source and a fiberoptic cable directed emitted light to the spectrometer. Excitationconditions are reported for each fluorescence spectrum (3000 msecirradiation at the indicated wavelength). Spectral data were collectedon a personal computer using the Ocean Optics software and overlays ofdifferent spectra were captured.

Sample preparation. SNAFL-1 was purchased as the free carboxylic acidfrom

Molecular Probes in a 1 mg vial. 0.3 mL of isopropyl alcohol and 0.7 mLof water was added to make a 1 mg/mL solution. A molecular weight (MW)of 426 for SNAFL-1 was used to calculate molarity (SNAFL-1=2.35 mM).4.25 uL of this solution was diluted to 1 mL with various 50 mMphosphate buffers to give 10 micromolar solutions with pH 6-10. 10micromolar solutions of SNAFL-2 (MW=460) were prepared in a similarfashion. EBIO-1 (MW=523), EBIO-2 (MW=627), and EBIO-3 (MW=489) wereobtained as bulk compounds from Epoch Biosciences. 1.6 mg of each solidpowder was carefully weighed out and dissolved in 3.2 mL of 40%isopropyl alcohol to give 0.5 mg/mL solutions. Emission spectra wereobtained for the various SNAFL and EBIO compounds at pH 6.0, 6.2, 6.4,6.6, 6.8, 7.0, 8.0 and 10.0. Examples of overlayed fluorescence emissionspectra are shown in FIG. 8 (SNAFL-1) and FIG. 9 (EBIO-3). All spectrashowed an isosbestic wavelength where all emission spectra overlap (SeeTable 1). This is a characteristic of ideal ratiometric performance withno competing fluorescent structures other than those shown above(lactone, naphthol, naphtholate).

pKa calculations. The pH at which two molecular species (tautomers) areequally represented is defined as the pKa. There are many variables thatcan affect pKa and methods for measurement are difficult since thestructures have overlapping absorbance. Therefore direct comparisonsfrom the literature can vary slightly. The calculations contained hereinare based on the assumption that, at pH 10, only the trianionicnaphtholate structure is present. The intensity of fluorescence at theemission maxima is divided by 2, and pH of the intersecting pH curve iscalculated by interpolation between the nearest 2 curves. The pKa of the2-chloro substituted EBIO compounds is significantly lower than theother analogs as shown in Table 1.

Example 4 The Preparation of Representative Fluorophore-ProteinConjugates: SNAFL-1/HSA

Human serum albumin (HSA) was purchased from Sigma (catalog # A-8763) as100 mg of lyophilized powder. SNAFL-1 NHS ester was purchased fromMolecular Probes as a mixture of the 5 and 6 isomers. A solution of 10mg (0.15 micromoles) of HSA in 1 mL of pH 8.56 sodium bicarbonate (0.1M) was prepared. A solution of 1 mL (1.91 micromoles) of the NHS esterin 0.1 mL of dimethylsulfoxide was prepared. 0.3 mL aliquots of the HSAsolution were transferred to a 1.6 mL Eppendorf tubes and variousoffering ratios of the NHS ester solution were added: tube 1, 11.8microliters (5 equivalents); tube 2, 23.6 microliters (10 equivalents),tube 3, 47.1 microliters (20 equivalents). The deep red solutions werevortexed and allowed to stand in the dark for at least one hour. The 5:1conjugate from tube 1 was purified by gel filtration chromatography on a0.5×20 cm column packed with Sephadex G-15 and pH 7.4 phosphate bufferedsaline (PBS). The conjugate was isolated as a fast moving red/orangeband in PBS and diluted to 0.75 mL with PBS to give a 4 mg/mL solutionof the protein conjugate. Most of the color eluted with the conjugate,but some small molecular weight (orange) impurities remained on top ofthe column. The column was clean enough to be re-used for purificationof the 10:1 and 20:1 conjugates. Each was eluted in PBS and diluted to0.75 mL to give ˜4 mg/mL solutions (60 micromolar based on HSAcomponent). The red solutions were stored refrigerated and protectedfrom light. 1 micromolar solutions of each SNAFL-1/HSA conjugate wereprepared and analyzed by UV-vis spectra using a Beckman DU640Bspectrometer. Each spectrum showed absorbance maxima at 490 and 521 nmat pH 7 as expected for the acid form of SNAFL-1 conjugates. Therelative absorbance showed the expected change in absorbance withdifferent SNAFL:HSA offering ratio. A 10 micromolar solution of SNAFL-1acid (obtained from Molecular Probes) at pH 7 was used as a standard tomore accurately determine the average loading of SNAFL-1 per each HSAconjugate preparation. Using this assay, the 5:1 conjugate had 4.1fluors/HSA, the 10:1 conjugate had 6.4 fluors/HSA, and the 20:1conjugate had 11.2 fluors/HSA.

Example 5 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates: SNAFL-1/HSA

Relative fluorescence of various SNAFL-1/HSA conjugates and SNAFL-1 freeacid were compared using an Ocean Optics USB2000 fiber opticspectrometer and a tungsten halogen light source (part number HL-2000FHSA). The light source was equipped with a linear variable filter thatallowed the wavelength and shape of the excitation beam to be adjusted.A cuvette holder (part number CUV-FL-DA) was directly attached to thelight source and a fiber optic cable directed emitted light to thespectrometer. Excitation conditions are reported for each fluorescencespectrum (3000 msec irradiation at the indicated wavelength). Spectraldata were collected on a personal computer using the Ocean Opticssoftware and overlays of different spectra were captured. A comparisonof various loading levels of SNAFL-1/HSA showed that 4.1 to 1.6 SNAFL-1molecules gave about the same fluorescent signal. Higher loading orlower loading conjugates gave lower signals.

Emission spectra were obtained for 10 micromolar solutions in potassiumphosphate buffer. Excitation was at 540 nm. Emission maximum at 620 nmwas observed for the base form of SNAFL-1 (pH 10). As expected,intensity of 620 nm fluorescence decreased as pH decreased. Anisosbestic point at 585 nm, where fluorescence remained constant at allpH, was observed. Response was good at about pH 8, but poor between pH6-7.

Spectra obtained for a 2.5 micromolar solution of a representativeSNAFL-1/HSA conjugate (1.6 SNAFL-1/HSA) showed improved pH response forpH 6-7 (see FIG. 11). The Ocean Optics halogen light source was equippedwith a 532 nm interference filter (Edmund Optics, Barrington, N.J.) andthis allowed the fluorescent isosbestic point at 572 nm for pH 6-7 to bedetected. Emission maximum at 620 nm was observed for the base form ofSNAFL-1 (see pH 10 curve). As expected, intensity of 620 nm fluorescencedecreased as pH decreased. In comparison to the free SNAFL-1 carboxylicacid (see FIG. 8) improved response for pH 6-7 was observed for the HSAconjugate. A red shift of the pH 8 and 10 curves from the isosbesticwavelength was observed, indicative of other competing molecularstructures involving the fluorescent species. This non-ideal behaviormay be eliminated by use of a longer linker structure or a morehydrophilic linker structure between the fluorescent dye and the HSAspacer.

Example 6 The Preparation of Representative Fluorophore-ProteinConjugates: EBIO-3/HSA

Method A. A 0.1 M stock solution of EDC (Sigma/Aldrich Chemical Co., St.Louis Mo.) was prepared by dissolving 6.2 mg of EDC in 0.2 mL of DMF and0.123 mL of 50 mM phosphate buffer (pH 5.8). 1.0 mg of EBIO-3 acid(Nanogen, Bothell, Wash.) was dissolved in 0.102 mL of DMF to give a 20mM solution. 3.0 mg (0.045 micromoles) of HSA (Sigma/Aldrich ChemicalCo., St. Louis, Mo.) was dissolved in 0.3 mL of pH 8.5 sodiumbicarbonate in each of two 1.7 mL Eppendorf tubes. 0.1M EDC (0.045 mL)was added to 20 mM EBIO-3 (0.045 mL, 0.9 micromoles) in a separateEppendorf tube and this was added to one of the HSA tubes to give an

EBIO-3:HSA offering ratio of 20:1. An offering ratio of 5:1 was used inthe other HSA tube by adding a premixed solution of 0.0225 mL of EDC(0.1 mM) and 0.0225 mL of EBIO-3 (20 mM). The homogeneous dark red HSAconjugate solutions were incubated at room temperature in the dark.After 21 hours, each of the HSA conjugates was purified on a G15Sephadex column as described above for the SNAFL conjugates

(Example 4). Some unreacted EBIO-3 acid remained at the top of thecolumn (especially for the 20:1 offering ratio), but was cleanlyseparated from the desired protein conjugate that eluted first as a pinkfraction in ˜0.5 mL of pH 7.4 buffer. Each of the purified conjugateswas diluted to 0.75 mL with pH 7.4 PBS to give 4 mg/mL solutions (0.06mM). The red solutions were stored refrigerated and protected fromlight.

1 micromolar solutions of each EBIO-3/HSA conjugate were prepared at pH7.4 and analyzed by UV-vis spectra using a Beckman DU640B spectrometer.The free EBIO-3 acid (10 micromolar) spectrum had absorbance maximum at534 nm, the 20:1 conjugate had absorbance at 538 nm and the 5:1conjugate had maximum at 545 nm. The spectra showed the expectedincrease in absorbance with increasing EBIO-3:HSA offering ratio. Usingthis EBIO-3 acid as a standard, the 20:1 conjugate had 5.07 EBIO-3:HSAand the 5:1 offering had 1.92 EBIO-3:HSA. The coupling efficiency wassomewhat lower than for the SNAFL/HSA conjugates of Example 4 (the 20:1conjugate had 11.2 fluors/HSA and the 5:1 offering had 4.1 fluors/HAS).The EDC coupling method was suitably efficient and reproducible.

Method B. A 0.1 M solution of EDC (Sigma/Aldrich Chemical Co., St.Louis, Mo.) is prepared by dissolving 6.0 mg of EDC in 0.194 mL of DMFand 0.118 mL of 50 mM PBS (pH 7.4). 3.0 mg of EBIO-3 acid (Nanogen,Bothell, Wash.) is dissolved in 0.306 mL of DMF to give a 20 mMsolution. The two solutions are combined in the EBIO-3 solutioncontainer and incubated at room temperature for one hour in the dark.75.0 mg (1 micromole) of liquid recombinant HSA (rHSA) from yeast (DeltaBiotechnology, Ltd., Nottingham, UK) is mixed with 7.5 mL of pH 8.5sodium bicarbonate in a 15 mL conical tube. The entire contents of theEBIO-3/EDC solution are combined with the rHSA solution and incubated atroom temperature in the dark for 15-20 hours. The rHSA/EBIO-3 conjugateis purified using the Amicon stirred ultrafiltration cell system and aYM10 membrane (Millipore, Bedford, Mass.). A 50 mM PBS (pH 7.4) is usedas the wash solution. After purification, the protein concentration ofthe conjugate is measured using the BCA™ Protein Assay (Pierce,Rockford, Ill.). An aliquot of the conjugate is diluted to 1 mg/ml with50 mM PBS (pH 7.4) based on its BCA determined protein concentration.The 1 mg/ml aliquot of conjugate, the last milliliter of PBS effluent,an aliquot of the 50 mM PBS (pH 7.4), and an aliquot of the EB3 Standard(15 mM EBIO-3 solution in DMF and 50 mM PBS (pH 7.4)) are analyzed viaan absorbance scan utilizing Bio-Tek's Synergy HT plate reader. The scanis taken on 300 microliters of each of the above mentioned samples in ablack, 96-well, clear, flat bottom plate, scanned from 450 nm to 650 nm.Their max peaks are recorded and used to determine purity and quality ofthe conjugate.

Example 7 The Fluorescent Properties of RepresentativeFluorophore-Protein Conjugates: EBIO-3/HSA

Fluorescence spectra were obtained for 2.5 micromolar solutions of thetwo EBIO-3/HSA conjugates prepared as described in Example 6 (Method A).The conjugates showed improved pH response for pH 6-7 (see FIG. 12 foroverlayed spectra for the 1.92:1 EBIO-3/HSA conjugate). The Ocean Opticshalogen light source was equipped with a 532 nm bandpass filter (EdmundOptics, Barrington N.J.) and this allowed the fluorescent isosbesticpoint at ˜565 nm for pH 6-7 to be detected. Emission maximum at 605 nmwas observed for the base form of SNAFL-1 (red trace, pH 10). Asexpected, intensity of 605 nm fluorescence decreased as pH decreased. Incomparison to the SNAFL-1/HSA conjugate (see FIG. 11) improved responsefor pH 6-7 was observed for the EBIO-3/HSA conjugate. A red shift of thepH 8 and 10 curves from the isosbestic wavelength was observed,indicative of other competing molecular structures involving thefluorescent species, but was of smaller magnitude than for theSNAFL-1/HSA conjugate.

Example 8 Immobilization of Representative Fluorophore-ProteinConjugates: SNAFL-1/HSA

Fluorophore-protein conjugates and fluorophore-carbohydrate conjugateswere immobilized on either nitrocellulose or capillary pore membranesusing the following general method. Fluorescein labeled dextrans with“fixable” lysine residues were obtained from Molecular Probes. Thesedextrans had a molecular weight of about 10,000 1.8 fluorophores perconjugate, and 2.2 lysines per conjugate and are sold under the tradename “Fluoro-Emerald”. Fluorescein labeled bovine serum albumin (BSA)was also obtained from Molecular Probes and had 4.5 fluors perconjugate. Various SNAFL-1/HSA conjugates were prepared as described inExample 4. Nitrocellulose membranes were obtained from Schleicher andSchuell under the trade name PROTRAN. Pore diameter was reported as 0.2microns. Capillary pore membranes made from polyester films wereobtained from Oxyphen in a variety of pore sizes. 0.1 micron and 1.0micron pore size membranes were successfully used to immobilizefluorescein dextrans. Fluorescein/dextran, fluorescein/BSA andSNAFL-1/HSA conjugates were all successfully immobilized and thefluorescent properties of the SNAFL-1/HSA conjugates were fullycharacterized as described as follows.

General Immobilization Method. SNAFL-1/HSA (2.5 SNAFL-1/HSA) on 0.1micron pore diameter Oxyphen Membrane Discs. Fluorescent HSA conjugateswith a 2.5:1 SNAFL-1:HSA offering ratio were prepared as described inExample 4 and diluted to provide concentrations of 0.05, 0.2, 1.0 and 4mg/mL in phosphate buffered saline (PBS) (pH 7.4). 5 microliter dropswere applied via a 20 microliter pipettor to the center of pre-punchedporous discs (1/4 inch diameter) that were laid on a bench top. Thespotted discs were allowed to air dry (about 30 minutes) and then placedin separate desiccators overnight. The dried discs were washed inseparate Eppendorf tubes with 2×1 mL of PBS and allowed to soakovernight in 1 mL of PBS. The washed discs were stable in PBS solution(no degradation after 30 days). Alternatively the discs could bere-dried in desiccators and stored dry. The wet or dry stored discs hadcomparable fluorescent properties. The discs had fluorescent signalsthat were proportional to the concentration of labeled macromoleculeapplied to each one as measured by the fluorescence assay described inExample 9.

Example 9 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates: SNAFL-1/HSA

Microwell assay of fluorescent macromolecular conjugates on porousmembrane discs using a fiber optic spectrometer. Fluorescent discsprepared as described in Example 8 were examined for fluorescentproperties using the Ocean Optics fiber optic spectrometer described inExample 5. The cuvette on the light source was replaced by a fiber opticreflectance probe which had 6 excitation fibers wrapped around a singlefiber that picks up the emitted light from the sample and sends it tothe spectrometer. The reflectance probe was threaded through a hole in a12×12×18 inch black box with a lid on the front. The probe was clampedinside under a 1 cm square opening that allowed the tip of the probe tobe positioned under a 96-well micro well plate (clear bottom blackplate). The probe was tilted at a 30 degree angle to reduce reflectedlight entering the probe tip. The fluorescent disc of interest wasplaced in the bottom of a well and covered with 300 microliters of theanalyte solution of interest. The excitation light source was turned onlong enough to position the disc of interest over the tip of thereflectance probe, then the shutter was closed and the plate was coveredwith another box to shield the disc from ambient light. Unless otherwisementioned, the Ocean Optics software was set to collect data with a 3000msec integration time and 3 averages. A dark spectrum was captured withthe shutter closed and used for all background subtracted readingsduring the assay. The shutter was then opened and fluorescent reading ofthe disc was started. The graphical display on the computer screen gavereal-time spectra after each 3000 msec integration time. After therequired 3 spectra were obtained (about 10 seconds) the graphicaldisplay showed only subtle changes. At this point a snapshot of thedisplayed spectrum was captured and saved to disc for future processing.The shutter was closed, and the next microwell experiment was set up.The same disc could be measured multiple times by exchanging the analytesolution in the microwells. Alternatively, different discs in differentwells could be measured by re-positioning the microwell plate over thereflectance probe.

Fluorescent loading of SNAFL-1/HSA immobilized on Oxyphen discs. Themicrowell assay described above was used to compare therelative-fluorescence of SNAFL-1/HSA on Oxyphen discs. The excitationfilters in the halogen light source were set to a wavelength of 532 nmand a “wide open” bandpass position to maximize sensitivity of theassay. Reflectance of the excitation beam back into the detector fiberwas significant, and the wavelength position of the filter was adjustedto provide a “minimum” at 620 nm where the fluorescence from the baseform of SNAFL-1/HSA is greatest. The various concentrations ofSNAFL-1/HSA described in Example 8 were examined in separate microwellsin pH 7 potassium phosphate buffer (50 mM) as described above. Thespectra showed the ability to distinguish relative fluorescenceintensity of 4, 1, 0.2 and 0.05 mg/mL membranes at pH 7. All had signalgreater than background.

Relative fluorescence intensity of various amounts of SNAFL-1/HSA(2.5:1) immobilized on porous Oxyphen discs at pH 7. The fluorescentintensity was measured at 620 nm, the fluorescent maximum of the baseform of the fluorophore. Excitation used a wide open setting on thehalogen lamp that efficiently excites both acid and base forms ofSNAFL-1. The reflected light from the source (unmodified disc) had thelowest intensity spectrum. The spectrum of the 0.05 mg/mL disc gave asmall increase in fluorescence intensity. The 0.2 mg/mL disc, 1 mg/mLdisc, and 4 mg/mL disc showed stepwise increases in fluorescenceintensity. The fluorescence spectra of two 30 day PBS soaked sample (1mg/mL) from a different batch of membranes were essentially the same andshowed that membrane loading was reproducible from batch to batch, andthat the SNAFL-1/HSA conjugates did not dissociate significantly fromthe disc surface in PBS solution.

pH Dependent fluorescence of SNAFL-1/HSA immobilized on Oxyphen discs.The 1 mg/mL SNAFL-1/HSA discs described above were examined for pHdependent response in the microwell assay. A single disc was examined inpotassium phosphate buffers of pH 4, 5, 6, 7, 8, 9, and 10. The datashowed that these membrane discs had a wide dynamic range of pHmeasurement, but had more sensitive response at pH>6. The time betweenbuffer exchanges was 5 min, and there was no significant change inspectra after additional equilibration time. This showed that theresponse time for even dramatic changes in the pH environment of theimmobilized SNAFL-1/HSA conjugates is rapid.

“Crossover assay” for fluorescence measurement of pH using SNAFL-1/HSAOxyphen discs. The microwell assay described above was used to examinethe fluorescent isosbestic properties of the discs. For this assay, theshutter assembly in Ocean Optics halogen light source (part numberHL-2000 FHSA) was removed, and two 532 nm bandpass filters (EdmundScientific) were inserted in the cavity using a special adaptor. Thisdramatically reduced the reflected background in the spectral region ofinterest (>550 nm). The data shown are for 4 mg/mL loading discsprepared with SNAFL:HSA (5:1) conjugate. The immobilized proteinconjugate showed unusual pH vs. fluorescence properties in comparison tothe solution phase data. Instead of a fluorescent isosbestic point at575 nm, there was a stepwise increase in the fluorescent intensity as pHincreased. The pH 10 spectrum showed the expected maximum at 620 nm, andcrossed the overlaid spectral curves obtained in pH 4, 6, 7 and 8buffers. These “crossover points” were used as the basis for a sensitiveassay to determine pH of the membrane environment. Three differentmembrane discs were examined using this assay format on three differentdays. The crossover points were reproducible within 2 nm.

The 4 mg/mL discs (3.6:1 SNAFL-1:HSA) showed stepwise increase in pH 10“crossover”. The crossover was at 579 nm for pH 4. Three discs/threedifferent days gave the same result ±2 nm. The crossover points were at592 nm (pH 6), 600 nm (pH 7a,b), and 611 nm (pH 8). The fluorescentmaximum at pH 10 was at 620 nm, similar to the solution phaseproperties.

Example 10 Immobilization of Representative Fluorophore-ProteinConjugates: EBIO-3/HSA

Spotting Immobilization Method. EBIO-3/HSA conjugate was prepared asdescribed in Example 6 at a ratio of 2:1. Nitrocellulose membranes wereobtained from Schleicher and Schuell under the trade name PROTRAN. Thediscs were treated in the same way as the general immobilization methoddescribed in Example 5 using a 4 mg/ml solution of EBIO-3/HSA.

Soaking Immobilization Method. EBIO-3/HSA conjugate was prepared asdescribed in Example 6 at a ratio of 2:1. Mixed ester nitrocellulose andcellulose acetate membranes were obtained from Millipore under theproduct series TF. The EBIO-3/HSA conjugate is diluted to 0.2 mg/ml and45 ml is added to a 9 cm disc of the membrane. The disc is agitatedovernight at room temperature and protected from light. The unboundconjugate is removed and the disc is washed with two 1 hour washes andone overnight wash all with agitation. The disc is then desiccated andstored dry. Smaller discs are punched from the 9 cm disc for studies.

Example 11 The Fluorescent Properties of Representative ImmobilizedFluorophore-Protein Conjugates: EBIO-3/HSA

Telescoping tubing insert assay of fluorescent macromolecular conjugateson porous membrane discs using a fiber optic spectrometer. Fluorescentdiscs prepared as described in Example 10 were examined to relate thefluorescent properties to the liquid phase pH using the Ocean Opticsfiber optic spectrometer described in Example 9 with the dual 532 nmfiltered (Edmund Scientific) halogen light source (part number HL-2000FHSA). A holder for a 5/32 inch membrane disc was crafted with 4 mm ODand 5 mm

OD polystyrene telescoping tubing and an angled 0.015 in thickpolystyrene window. The angled window was placed so that it held themembrane disc at a 60 degree angle relative to the tubing axis. Thisallows the fiber optic probe to be placed in one end of the tubing andinterrogate the disc on the other side of the window which is contactwith a liquid of a certain pH. Buffers of known pH values were placed incontact with the telescoping tubing inserts and discs made by thespotting immobilization method in Example 10 and fluorescent emissionsrecorded with the Ocean Optics software set to collect data with a 1000msec integration time and 3 averages.

For liquids with unknown pH values, a stirred and light protected vesselcontaining 5 telescoping tubing inserts and discs made by the soakingimmobilization method in Example 10, 50 mL of buffer or plasma, and acalibrated pH electrode (ROSS electrode/Orion 720a meter) was used tostudy the pH and fluorescent response of the fluorescent discs. Drops of1 N HCl or 1 M NaOH were added to create a range of pHs from liquidsstudied. Fluorescent spectra were collected through Ocean Optics macrosin Excel set to read for 1000 msec integration time and three averages.The spectra were analyzed using the modeled bandpass filters andratiometric method in Excel to obtain calibration curves for PBS,platelet poor plasma and platelet rich plasma.

Injection molded insert PVC tube assay of fluorescent macromolecularconjugates on porous membrane discs using a custom optimizedfluorescence based pH detector. Injection molded polycarbonate partswere fashioned to fix the fluorescent discs to the fluorescence pHdetector probe as pictured in FIG. 4. Membranes were prepared asdescribed in the soaking immobilization method in Example 10 andassembled into the plastic insert. A 1 in long and 3/16 in ID PVC tubewas placed on the spike end of the insert such that 250 ul of liquid wasplaced in the tube and covered with parafilm to slow carbon dioxidedesorption. A fluorescent measurement of the first and secondwavelengths was taken and then the pH was read by a blood gas analyzer(Bayer 348). The pH of plasma samples were adjusted by acid and baseadditions as in the telescoping tubing insert assay to create the rangeof pH data.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A method for measuring the pH of a sample, comprising: (a)irradiating a fluorescent species immobilized on a substrate withexcitation light emanating from a probe physically isolated from thefluorescent species immobilized on the substrate, wherein thefluorescent species immobilized on the substrate is in liquidcommunication with a sample, wherein the excitation light has awavelength sufficient to effect fluorescent emission from thefluorescent species, wherein the fluorescent species exhibits a firstemission intensity at a first emission wavelength and a second emissionintensity at a second emission wavelength, the ratio of the first andsecond emission intensities being dependent on pH; and (b) measuring thefirst and second emission intensities to determined the pH of thesample.
 2. The method of claim 1, wherein the probe is physicallyisolated from the fluorescent species immobilized on the substrate by awindow transparent to the excitation light and the fluorescent emission.3. The method of claim 1, wherein the probe comprises one or moreoptical fibers.
 4. The method of claim 1, wherein the fluorescentspecies is selected from the group consisting of a naphthofluoresceincompound and a seminaphthorhodamine compound.
 5. The method of claim 1,wherein the naphthofluorescein compound is selected from the groupconsisting of a seminaphthofluorescein compound and acarboxynaphthofluorescein compound.
 6. The method of claim 1, whereinthe fluorescent species is a seminaphthofluorescein compound selectedfrom the group consisting of 5′ (and6′)-carboxy-3,10-dihydroxy-spiro[7H-benzo[c]xanthene-7,1′(3′H)-isobenzofuran]-3′-oneand2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.
 7. The method of claim 1, wherein the fluorescent speciesimmobilized on a substrate comprises a conjugate of a fluorescentspecies and a macromolecule.
 8. The method of claim 7, wherein themacromolecule is an albumin.
 9. The method of claim 1, wherein thefluorescent species immobilized on a substrate comprises aseminaphthofluorescein/human serum albumin conjugate.
 10. The method ofclaim 1, wherein the sample comprises blood or a blood product.
 11. Themethod of claim 1, wherein the sample is contained within a sealedvessel.
 12. A method for measuring the pH of a sample, comprising:  (a)irradiating a fluorescent species in communication with a sample withexcitation light having a wavelength sufficient to effect fluorescentemission from the fluorescent species, wherein the fluorescent speciesexhibits a first emission intensity at a first emission wavelength and asecond emission intensity at a second emission wavelength, the ratio ofthe first and second emission intensities being dependent on pH, andwherein the fluorescent species comprises a 2-haloseminaphthofluorescein; and  (b) measuring the first and second emissionintensities to determined the pH of the sample.
 13. The method of claim12, wherein the 2-halo seminaphthofluorescein is a 2-chloroseminaphthofluorescein.
 14. The method of claim 12, wherein the 2-haloseminaphthofluorescein is2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid.
 15. The method of claim 12, wherein the fluorescent speciescomprises a conjugate of a 2-halo seminaphthofluorescein and amacromolecule.
 16. The method of claim 15, wherein the macromolecule isan albumin.
 17. The method of claim 15, wherein the macromolecule is ahuman serum albumin.
 18. The method of claim 15, wherein the conjugateis a2-(2-chloro-3-hydroxy-9-carboxyethyl-10-oxo-10H-benzo[c]xanthen-7-yl)benzoicacid/human serum albumin conjugate.
 19. The method of claim 12, whereinthe sample comprises blood or a blood product.